Overexpression of Rho-GTPases and Rho-GEFs has been described in various types of human tumors [18
], and in some cases overexpression is associated with tumor progression or poor prognosis [22
]. However, little is known about the role of Rho-GTPases in cervical carcinogenesis. Here, using immunohistochemistry, we show that the immunoreactivity of the GTPases Rac1 and RhoA, and the Rho GEFs Tiam1 and beta-Pix, is increased in SILs, compared to cervical epithelium without SIL. Interestingly, we found that Rac1 is present in the nucleus of a subset of L-SIL and H-SIL, but not in samples without SIL. In agreement with these findings, we observed nuclear localization of Rac1 in cancer derived C33A and SiHa cells but not in non-tumorigenic HaCat cells.
Rac1 has a nuclear localization signal (NLS) [24
], and it has been recently shown that the importin Karyopherin alpha 2 (KPNA2) mediates Rac1 nuclear import trough the interaction with its NLS, and that KPNA2-mediated nuclear import of Rac1 requires Rac1 activation [25
]. Here we show that the nuclear localization of Rac1 in C33A and SiHa cells is not affected by treatment with the Rac1 inhibitor NSC23766. These data indicate that in these cells, the presence of Rac1 in the nucleus is not dependent on its activation. Michaelson et al. (2010) showed that Rac1 translocates to the nucleus during the G2 phase of the cell cycle, and that targeting an active form of Rac1 to the nucleus promotes cell proliferation [26
]. We found that chemical inhibition of Rac1 reduces the proliferation of cervical cancer cell lines C33A and SiHa, as well as that of non-tumorigenic HaCat cells. In HaCat cells, in which Rac1 is localized to the cytoplasm, chemical inhibition of Rac1 may reduce its nuclear translocation during the G2 phase of the cell cycle, resulting in a reduction in cell proliferation. Buongiorno et al. (2008) showed that an inactive form of Rac1 is present in the nucleus of colorectal cancer cells, where it associates with the transcription factor TCF-4 [27
]. Interestingly, these authors demonstrated that activation of the Wnt signaling pathway induced the nuclear translocation of Tiam1, a Rac1-specific activator, in a complex with beta-catenin, and that once in the nucleus a beta-catenin/Tiam1/TCF4/Rac1 complex can be formed, resulting in the activation of Rac1 and transcriptional activation of Wnt target genes [27
]. Activation of the Wnt signaling pathway plays an important role during cervical cancer progression [28
]; therefore nuclear Rac1 may cooperate with this pathway to stimulate proliferation of cervical cancer cells. We found that chemical inhibition Rac1 in C33A and SiHa cells, in which Rac1 localizes both to the cytoplasm and the nucleus, impairs proliferation without affecting Rac1 nuclear localization. In these cells, inactivation of the nuclear pool of Rac1 may impair the interaction of Rac1 with nuclear proteins such as TCF4 and beta-catenin, resulting in a reduction in the expression of proliferation-related genes and therefore the reduction in cell proliferation. However, Rac1 can also regulate proliferation trough the activation of cytoplasmic signaling pathways such as NF-kB [30
], MAPK [31
], Jak/Stat [32
] and Wnt [33
] pathways. Therefore, it is possible that inhibition of the cytoplasmic pool of Rac1 in both cervical cancer-derived and non-tumorigenic cells may result in a reduction of cell proliferation, independently of Rac1 nuclear functions. Altogether, these data suggest that nuclear Rac1 may play an important role in regulating cell proliferation and gene expression in cervical cells, and that the presence of Rac1 in the nucleus of cervical epithelial cells from pre-malignant lesions may contribute to cancer progression.
In our study, we observed overexpression of Rac1, RhoA and Tiam1 in L-SIL and H-SIL, and beta-Pix in H-SIL, when compared with epithelia without SIL. In vitro experiments in HeLa cells demonstrate that Rac1 [34
] and Rho [35
] activation is required for cell growth and migration. Similarly, experiments in CaSki cells showed that inhibition of migration and invasion by the anticancer agent JOTO1007, is associated with a reduction in the expression of RhoA and the Rho downstream effector ROCK-1 [36
]. Moreover, experimental evidences indicate that Rho GTPases play a role in cellular transformation. It has been shown that Rac1 and its activator Tiam1 are required for Src-induced transformation [37
]. Similarly, it has been demonstrated that Rac1 and Cdc42 are necessary for H-Ras-induced transformation, although overexpression of constitutively active forms of Rac1 or Cdc42 is not sufficient for cellular transformation [38
]. It has also been shown that RhoA overexpression can induce pre-neoplastic transformation of primary mammary epithelial cells [39
]. These data suggest that overexpression of Rho GTPases in SILs may cooperate with other signaling pathways to promote tumor progression.
We found that the increased immunoreactivity of Rac1, RhoA and beta-Pix correlates with the histological diagnosis but not with HR-HPV infection. In contrast, Tiam1 immunoreactivity was associated with both histological diagnosis and HR-HPV infection. These observations suggest that altered expression of Tiam1, but not that of Rac1, RhoA and beta-Pix may be dependent of HR-HPV infection. However, further studies are needed in order to determine if increase levels of Rho proteins and their GEFs is induced directly by HPV oncoproteins or is the result of a secondary event related to the progression of the malignancy. Our data indicate that nuclear expression of Rac1 in cervical lesions may be independent of HR-HPV infection as not all HR-HPV positive samples have nuclear staining for Rac1. Moreover, both HPV-negative and HPV-positive cervical cancer derived cells have nuclear staining for Rac1. However, as mentioned above, it is possible that infection with other HPV types not detected by ISH technique we used in this work may affect the subcellular localization of Rac1. Moreover, ISH does not allow us to identify which HR-HPV type is present in the samples, and it is possible that infection with some HR-HPVs such as HVP16 and HPV18 will have a more dramatic effect on the expression of these proteins. This could be of particular relevance for our study population, as in a recent study performed on women from Guerrero state in the south of Mexico, Illades-Aguiar et al., (2010) reported that whereas HPV16 is the most frequent HPV type present in women with cervical cancer, the most frequent type in women with L-SIL was HPV33 [40
]. We also found moderate-strong reactivity for the five proteins in samples without SIL (Table ). Recent evidences demonstrate infection with HR-HPVs in patients without SILs [40
]. It is possible that some of the samples without SIL that showed moderate-strong reactivity are positive to HR-HPV. As mentioned above, we used ISH for the detection of HR-HPV infection. However this method has limitations as it detects only a subset of HR-HPV types. Further studies using more sensitive techniques such as PCR-RFLP or sequencing for the detection and typing of HPV infection will be required to answer to this concern. Finally, we could not determine HR-HPV infection in a subset of samples. Further investigation is required to determine the possible association between the overexpression of Rho-GTPases and HR-HPV infection.
One of the limitations in our study is that expression of the analyzed proteins in cervical biopsies was studied only by immunochemistry. Further studies using Western blotting, as well as analysis of a larger number of samples are required.